Modulation of epidermal growth factor receptor ligands

ABSTRACT

The present invention relates to a method for modulating the expression and/or activity of an epidermal growth factor receptor (EGFR) ligand in a cell or tissue, the method comprising contacting the cell or tissue with a miR-7 miRNA, a pre-cursor or variant thereof, a miRNA comprising a seed region comprising the sequence GGAAGA, or an antagonist of any such miRNA.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of Australian Provisional PatentApplication No. 2009905758 filed 24 Nov. 2009, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to methods for modulating theactivity and/or expression of epidermal growth factor receptor (EGFR)ligands such as transforming growth factor-alpha (TGFα). In particular,the present invention relates to methods for modulating EGFR ligandexpression and/or activity utilizing miRNA and to methods for treatingconditions associated with dysregulated expression and/or activity ofEGFR ligands.

BACKGROUND

The epidermal growth factor (EGF) family includes EGF, transforminggrowth factor-alpha (TGFα), heparin binding EGF-like growth factor(HB-EGF), amphiregulin (AR), epiregulin (EPR), betacellulin (BIC),epigen and the neuregulins (NRG)-1, NRG-2, NRG-3 and NRG-4. Members ofthe EGF family are ligands for the epidermal growth factor receptor(EGFR), a ligand activated receptor tyrosine kinase and member of theErbB receptor family. EGFR ligands are important in many cellularsignalling pathways and dysregulation of EGFR ligands is apparent in anumber of diseases. For example, in non-small-cell lung cancer,increased plasma TGFα is associated with erlotinib resistance andincreased amphiregulin is an indicator of poor prognosis. TGFα isinvolved in the stimulation and control of cell proliferation anddifferentiation and is produced in normal tissues by macrophages,hepatocytes, platelets and keratinocytes. TGFα is also produced by anumber of carcinomas and upregulation of expression of TGFα has beenfound in many forms of cancer accordingly TGFα is a target of anticancertherapies.

EGFR is a target for anti-cancer therapies as it is over expressed in alarge number of cancers. For example, more than 80% of all head and neckcancers (HNCs) overexpress EGFR. Signalling from EGFR results inactivation of downstream phosphoinositide 3-kinase (PI3K)/Akt andRas/Raf/MAPK pathways that promote tumour proliferation, invasion,metastasis, angiogenesis and apoptosis inhibition which all contributeto cancer progression and poor patient prognosis. However, clinicaltrials of tyrosine kinase inhibitors targeting EGFR, including gefitiniband erlotinib and the monoclonal antibody cetuximab, have shown onlylimited effectiveness in a range of cancers including HNCs. Similarly,anti-Akt agents also have limited therapeutic effectiveness.

microRNAs (miRNAs) are an abundant class of highly conserved, small(typically 21-25 nucleotides) endogenous non-protein-coding RNAs thatnegatively regulate gene expression. miRNAs bind specific3′-untranslated regions (3′-UTRs) within messenger RNAs (mRNA) to inducemRNA cleavage or translational repression. Individual miRNAs typicallybind incompletely to their cognate target messenger RNA (mRNA) and aunique miRNA may regulate the expression of multiple genes.

miRNAs are generated from RNA precursors (pri-miRNAs) that usuallycontain several hundred nucleotides transcribed from regions ofnon-coding DNA. Pri-miRNAs are processed in the nucleus by RNase IIIendonuclease to form stem-loop precursors (pre-miRNAs) of approximately70 nucleotides. Pre-miRNAs are actively transported into the cytoplasmwhere they are further processed into short RNA duplexes, typically of21-23 bp. The functional miRNA strand dissociates from its complementarynon-functional strand and locates within theRNA-induced-silencing-complex (RISC). (Alternatively, RISC can directlyload pre-miRNA hairpin structures.) miRNAs bind the 3′UTRs of targetmRNAs and important in this binding is a so-called ‘seed’ region ofapproximately 6-7 nucleotides near the 5′ end of the miRNA (typicallynucleotide positions 2 to 8). The role of the 3′ end is less clear.miRNA-induced regulation of gene expression is typically achieved bytranslational repression, either degrading proteins as they emerge fromribosomes or ‘freezing’ ribosomes, and/or promoting the movement oftarget mRNAs into sites of RNA destruction.

miRNAs are crucial to many normal cellular functions and are involved inprocesses such as stem cell division, embryonic development, cellulardifferentiation, inflammation and immunity. Increasingly, specificmiRNAs, and expression patterns and altered regulation of expression ofindividual miRNAs, are also being implicated in a variety of diseaseconditions, including cancer. Some miRNAs are altered in cancer and mayact as tumour suppressors or oncogenes. For example, let-7d (a member ofthe let-7 family of miRNAs) regulates RAS oncogene expression in normalhead and neck tissue although let-7d expression is reduced in many headand neck cancers causing upregulation of RAS expression, increasedtumour growth and reduced patient survival. In contrast, miR-184expression is upregulated in tongue squamous cell carcinoma, leading toincreased expression of the oncogene c-Myc, increased cell proliferationand tumour growth.

SUMMARY

In a first aspect the present invention provides a method for modulatingthe expression and/or activity of an epidermal growth factor receptor(EGFR) ligand in a cell or tissue, the method comprising contacting thecell or tissue with a miR-7 miRNA, a precursor or variant thereof, amiRNA comprising a seed region comprising the sequence GGAAGA, or anantagonist of any such miRNA.

The miR-7 miRNA may be hsa-miR-7 and may comprise the nucleotidesequence set forth in SEQ ID NO:1. The miR-7 miRNA precursor may beselected from hsa-miR-7-1, hsa-miR-7-2 and hsa-miR-7-3, and may comprisea sequence as set forth in any one of SEQ ID Nos:2 to 4.

Typically contacting the cell or tissue with the miRNA reduces orinhibits the expression and/or activity of the EGFR ligand. Contactingthe cell or tissue with an antagonist of the miRNA may increase theexpression and/or activity of the ligand.

Typically the 3′ untranslated region of the mRNA encoding the EGFRligand comprises one or more miRNA binding sites. Typically the miRNAbinds to one or more of the sites. The binding sites may comprisesequences as set forth in any of SEQ ID Nos:6 to 11, or variantsthereof.

The EGFR ligand may be selected from TGFα and HB-EGF. In a particularembodiment the EGFR ligand is TGFα. The mRNA encoding the TGFα maycomprise a 3′ untranslated region comprising the sequence set forth inSEQ ID NO:12, or a variant thereof.

The miRNA or antagonist thereof may be contacted with the cell or tissuein vivo or ex vivo. The subject containing the cell or tissue, or fromwhich the cell or tissue is derived, may suffer from, be predisposed to,or otherwise at risk of developing a disease or condition associatedwith dysregulated expression or activity of the EGFR ligand. The diseaseor condition may be associated with upregulated or elevated expressionor activity of the EGFR ligand. The disease or condition may be acancer. The cancer may be, for example, a head and neck cancer, agioblastoma, pancreatic cancer, colon cancer, lung cancer including nonsmall cell lung cancer, prostate cancer, breast cancer, liver cancer,neuroblastoma or melanoma.

In a second aspect the present invention provides a method formodulating the expression and/or activity of an epidermal growth factorreceptor (EGFR) ligand in a cell or tissue, the method comprisingcontacting the cell or tissue with an agent capable of stimulating orenhancing the expression or activity of a miR-7 miRNA, a precursor orvariant thereof, or a miRNA comprising a seed region comprising thesequence GGAAGA, whereby the miRNA the expression or activity of whichis stimulated or enhanced modulates the expression and/or activity ofthe EGFR ligand.

In a third aspect the present invention provides a method for treating adisease or condition associated with dysregulated expression or activityof an EGFR ligand in a subject, comprising administering to the subjectan effective amount of a miR-7 miRNA, a precursor or variant thereof, amiRNA comprising a seed region comprising the sequence GGAAGA, or anantagonist of any such miRNA, whereby the miRNA modulates the expressionor activity of the EGFR ligand.

In a particular embodiment the disease or condition is associated withupregulated or elevated expression or activity of the EGFR ligand andthe subject is administered an effective amount of a miR-7 miRNA, aprecursor or variant thereof, a miRNA comprising a seed regioncomprising the sequence GGAAGA.

The miR-7 miRNA may comprise the nucleotide sequence as set forth in SEQID NO:1. The miR-7 miRNA precursor may comprise a sequence as set forthin any one of SEQ ID Nos:2 to 4.

Typically contacting the cell or tissue with the miRNA reduces orinhibits the expression or activity of the EGFR ligand. Contacting thecell or tissue with an antagonist of the miRNA may increase theexpression or activity of the ligand.

Typically the 3′ untranslated region of the mRNA encoding the EGFRligand comprises one or more miRNA binding sites. Typically the miRNAbinds to one or more of the sites. The binding sites may comprisesequences set forth in any of SEQ ID Nos:6 to 11, or variants thereof.

The EGFR ligand may be selected from TGFα and HB-EGF. In a particularembodiment the EGFR ligand is TGFα. The mRNA encoding the TGFα maycomprise a 3′ untranslated region comprising the sequence set forth inSEQ ID NO:12, or a variant thereof.

The disease or condition may be a cancer. The cancer may be, forexample, a head and neck cancer, a glioblastoma, pancreatic cancer,colon cancer, lung cancer including non small cell lung cancer, prostatecancer, breast cancer, liver cancer, neuroblastoma or melanoma.

The miRNA may be co-administered with one or more additional therapeuticagents suitable for the treatment of the disease or condition. Inparticular embodiments the additional therapeutic agent is a tyrosinekinase inhibitor or monoclonal antibody, such as an inhibitor of EGFR.Co-administration may comprise simultaneous or sequential administrationof the miRNA and the one or more additional agents. For simultaneousadministration the miRNA and the one or more additional agents may beformulated in a single pharmaceutical composition together withpharmaceutically acceptable carriers, excipients or adjuvants.

In a fourth aspect the present invention provides a method for treatinga disease or condition associated with upregulated or elevatedexpression or activity of an EGFR ligand in a subject, comprisingadministering to the subject an effective amount of an agent capable ofstimulating or enhancing the expression or activity of a miR-7 miRNA, aprecursor or variant thereof, or a miRNA comprising a seed regioncomprising the sequence GGAAGA, whereby the miRNA the expression oractivity of which is stimulated or enhanced modulates the expressionand/or activity of the EGFR ligand.

A fifth aspect of the present invention provides the use of a miR-7miRNA, a precursor or variant thereof, or a miRNA comprising a seedregion comprising the sequence GGAAGA for the manufacture of amedicament for the treatment of a disease, or condition associated withupregulated or elevated expression or activity of an EGFR ligand,whereby the miRNA modulates the expression or activity of the EGFRligand.

A sixth aspect of the invention provides the use of an agent capable ofstimulating or enhancing the expression or activity of a miR-7 miRNA, aprecursor or variant thereof, or a miRNA comprising a seed regioncomprising the sequence GGAAGA, for the manufacture of a medicament forthe treatment of a disease or condition associated with upregulated orelevated expression or activity of an EGFR ligand, whereby the miRNA theexpression or activity of which is stimulated or enhanced modulates theexpression and/or activity of the EGFR ligand.

A seventh aspect of the present invention provides a method forpreventing or reducing tumour growth, cancer metastasis or reoccurrencein a subject, wherein the tumour or cancer is associated withupregulated or elevated expression or activity of an EGFR ligand, themethod comprising administering to the subject an effective amount of amiR-7 miRNA, a precursor or variant thereof, or a miRNA comprising aseed region comprising the sequence GGAAGA, whereby the miRNA modulatesthe expression or activity of the EGFR ligand.

An eighth aspect of the present invention provides a method forpreventing or reducing tumour growth, cancer metastasis or reoccurrencein a subject, wherein the tumour or cancer is associated withupregulated or elevated expression or activity of an EGFR ligand, themethod comprising administering to the subject an effective amount of anagent capable of stimulating or enhancing the expression or activity ofa miR-7 miRNA, a precursor or variant thereof, or a miRNA comprising aseed region comprising the sequence GGAAGA, whereby the miRNA theexpression or activity of which is stimulated or enhanced modulates theexpression and/or activity of the EGFR ligand.

A ninth aspect of the present invention provides a method for evaluatingthe efficacy of a treatment regime in a subject suffering from a diseaseor condition associated with dysregulated expression or activity of anEGFR ligand, the method comprising:

(a) treating the subject with a miR-7 miRNA, a precursor or variantthereof, a miRNA comprising a seed region comprising the sequenceGGAAGA, or an antagonist of any such miRNA, for a period sufficient toevaluate the efficacy of the regime;

(b) obtaining a biological sample from the subject;

(c) determining the level of expression and/or activity of the EGFRligand in the sample;

(d) repeating steps (b) and (c) at least once over a period of time oftreatment; and

(e) determining whether the expression and/or activity of the EGFRligand change over the period of time,

wherein a change in the level of expression and/or activity of the EGFRligand is indicative of the efficacy of the treatment regime.

In particular embodiments the EGFR ligand is TGFα.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are described and exemplified herein, byway of non-limiting example only, with reference to the followingfigures.

FIG. 1 shows the experimental validation of genes down-regulated bymiR-7 by cDNA analysis. Quantitative RT-PCR of RAF1 and PAK1 mRNAexpression in HN5 cells 24 h after transfection with 30 nM miR-7 ormiR-NC precursor. RAF1 and PAK1 mRNA expression was normalised to GAPDHmRNA expression and is shown as a ratio of miR-NC-transfected cells(±SD) using the 2^(−ΔΔCT) method. Bars represent mean mRNA expression(±SD) compared to miR-NC. Data representative of a single experiment.*** and ** indicate a significant difference from miR-NC treated cells(p<0.001 and p<0.01 respectively).

FIG. 2 shows a model of miR-7 (SEQ ID NO:1) action on EGFR signalling inHNC cells. Schematic model using cDNA microarray data showing miR-7regulation of EGFR signalling via multiple targets. Genes found to bedown-regulated by miR-7 as per the cDNA microarray are outlined in bold.

FIG. 3 shows normalied relative expression levels for TGFα mRNA asdetermined by quantitative RT-PCR in HN5 cells (A) and FaDu cells (B) inthe presence of 30 nM miR-7 (SEQ ID NO:1) or miR-NC. Data arerepresentative of three independent experiments. *** indicates asignificant difference from miR-NC treated cells (p<0.001).

FIG. 4A shows luciferase reporter assays to verify activity of miR-7upon the consensus miR-7 target site. HN5 cells were transfected withconsensus miR-7 target site firefly luciferase plasmid and 1 nM miR-7 ormiR-NC precursor. Relative luciferase expression (firefly normalised toRenilla) values are shown as a ratio of vehicle (Lipofectamine 2000, LF)only. Bars represent standard deviation (SD). Data are representative ofa single experiment. *** indicates a significant difference from vehicle(Lipofectamine 2000, LF)-treated reporter vector (p<0.001).

FIG. 4B shows luciferase reporter assays to verify activity of miR-7upon a miR-7 target site within the full-length wild-type TGFα 3′-UTR 24h after transfection. HN5 cells were transfected with wild-type TGFαmiR-7 target site number 5 3′-UTR firefly luciferase plasmid and 0.5 nMmiR-7 or miR-NC precursor. Relative luciferase expression (fireflynormalised to Renilla) values are shown as a ratio of vehicle(Lipofectamine 2000, LF) only. Bars represent standard deviation (SD).Data are representative of a single experiment. ** indicates asignificant difference from vehicle (Lipofectamine 2000, LF)-treatedreporter vector (p<0.01).

A listing of nucleotide sequences corresponding to the sequenceidentifiers referred to in the specification is provided. The nucleotidesequences of mature human miR-7, human miR-7 precursors and seed regionare set forth in SEQ ID Nos:1 to 5. The sequence of a consensus miR-7binding site is provided in SEQ ID NO:6. Predicted miR-7 binding siteswithin the human TGFα 3′ untranslated region are set forth in SEQ IDNos:7 to 11, while the 3′ untranslated region of human TGFα 3′ isprovided in SEQ ID NO:12. SEQ ID Nos:13 to 23 provide sequences ofoligonucleotides used in the present study as exemplified herein.

DEFINITIONS

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid molecule”includes a plurality of nucleic acid molecules, and a reference to “acell” is a reference to one or more cells, and so forth.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

In the context of this specification, the term “activity” as it pertainsto a protein, polypeptide or polynucleotide means any cellular function,action, effect or influence exerted by the protein, polypeptide orpolynucleotide, either by a nucleic acid sequence or fragment thereof,or by the protein or polypeptide itself or any fragment thereof.

It will be understood that as used herein the term “expression” mayrefer to expression of a polypeptide or protein, or to expression of apolynucleotide or gene, depending on the context. The polynucleotide maybe coding or non-coding (e.g. miRNA). Expression of a polynucleotide maybe determined, for example, by measuring the production of RNAtranscript levels. Expression of a protein or polypeptide may bedetermined, for example, by immunoassay using an antibody(ies) that bindwith the polypeptide.

The terms “modulate,” “modulation,” “modulating”, “modulator” andgrammatical equivalents as used herein refer to the act of, and toagents described herein which are capable of, affecting directly orindirectly the activity and/or expression level of EGF receptor (EGFR)ligands such that the activity or expression is altered when compared to“wild-type” activity or expression i.e. activity or expression beforecontacting with an agent of the present invention. The term “indirectly”in the context of modulation of EGFR ligand activity or expressionrefers to the mode of action of an agent, wherein the effect is mediatedvia an intermediary molecule rather than through direct contact withEGFR ligand. In contrast, the term “directly” in the context ofmodulation of EGFR ligand activity or expression refers to an agent thatinteracts with the EGFR ligand or its mRNA by, for example, binding tothe 3′-UTR.

In the context of this specification, the term “antagonist” refers toany agent capable of blocking or inhibiting the expression and/oractivity of an EGFR ligand. Thus, the antagonist may operate to preventtranscription or post-transcriptional processing of the EGFR ligand orotherwise inhibit the activity of the EGFR ligand in any way, via eitherdirect or indirect action. The antagonist may for example be nucleicacid, peptide, any other suitable chemical compound or molecule or anycombination of these. Additionally, it will be understood that inindirectly impairing the activity of the EGFR ligand, the antagonist mayalter the activity and/or expression of other cellular molecules whichmay in turn act as regulators of the activity and/or expression ofactivity of the EGFR ligand itself. Similarly, the antagonist may alterthe activity of molecules which are themselves subject to regulation ormodulation by the EGFR ligand.

As used herein the term “oligonucleotide” refers to a single-strandedsequence of ribonucleotide or deoxyribonucleotide bases, known analoguesof natural nucleotides, or mixtures thereof. An “oligonucleotide”comprises a nucleic-acid based molecule including DNA, RNA, PNA, LNA orany combination thereof. An oligonucleotide that predominantly comprisesribonucleotide bases, natural or non-natural, may be referred to as anRNA oligonucleotide. Oligonucleotides are typically short (for exampleless than 50 nucleotides in length) sequences that may be prepared byany suitable method, including, for example, direct chemical synthesisor cloning and restriction of appropriate sequences. “Antisenseoligonucleotides” are oligonucleotides complementary to a specific DNAor RNA sequence. Typically in the context of the present invention anantisense oligonucleotide is an RNA oligonucleotide complementary to aspecific miRNA. The antisense oligonucleotide binds to and silences orrepresses, partially of fully, the activity of its complementary miRNA.Not all bases in an antisense oligonucleotide need be complementary tothe ‘target’ or miRNA sequence; the oligonucleotide need only containsufficient complementary bases to enable the oligonucleotide torecognise the target. An oligonucleotide may also include additionalbases. The antisense oligonucleotide sequence may be an unmodifiedribonucleotide sequence or may be chemically modified or conjugated by avariety of means as described herein.

The term “polynucleotide” as used herein refers to a single- ordouble-stranded polymer of deoxyribonucleotide, ribonucleotide bases orknown analogues of natural nucleotides, or mixtures thereof. A“polynucleotide” comprises a nucleic-acid based molecule including DNA,RNA, PNA, LNA or any combination thereof. The term includes reference tothe specified sequence as well as to the sequence complimentary thereto,unless otherwise indicated. Polynucleotides may be chemically modifiedby a variety of means known to those skilled in the art. Thus a“polynucleotide” comprises a nucleic-acid based molecule including DNA,RNA, PNA, LNA or any combination thereof.

The term “sequence identity” or “percentage of sequence identity” may bedetermined by comparing two optimally aligned sequences or subsequencesover a comparison window or span, wherein the portion of thepolynucleotide sequence in the comparison window may optionally compriseadditions or deletions (i.e., gaps) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences.

As used herein the term “associated with” when used in the context of adisease or condition means that the disease or condition may resultfrom, result in, be characterised by, or otherwise associated withabnormal EGF ligand levels. Thus, the association between the disease orcondition and the abnormal EGF ligand levels may be direct or indirectand may be temporally separated.

As used herein the terms “treating” and “treatment” and grammaticalequivalents refer to any and all uses which remedy a condition orsymptoms, prevent the establishment of a condition or disease, orotherwise prevent, hinder, retard, or reverse the progression of acondition or disease or other undesirable symptoms in any waywhatsoever. Thus the term “treating” is to be considered in its broadestcontext. For example, treatment does not necessarily imply that apatient is treated until total recovery. In conditions which display orare characterized by multiple symptoms, the treatment need notnecessarily remedy, prevent, hinder, retard, or reverse all of saidsymptoms, but may prevent, hinder, retard, or reverse one or more ofsaid symptoms.

As used herein the term “effective amount” includes within its meaning anon-toxic but sufficient amount or dose of an agent or compound toprovide the desired effect. The exact amount or dose required will varyfrom subject to subject depending on factors such as the species beingtreated, the age and general condition of the subject, the severity ofthe condition being treated, the particular agent being administered andthe mode of administration and so forth. Thus, it is not possible tospecify an exact “effective amount”. However, for any given case, anappropriate “effective amount” may be determined by one of ordinaryskill in the art using only routine experimentation.

The term “subject” as used herein refers to mammals and includes humans,primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys),laboratory test animals (e.g. mice, rabbits, rats, guinea pigs),performance and show animals (e.g. horses, livestock, dogs, cats),companion animals (e.g. dogs, cats) and captive wild animals.Preferably, the mammal is human or a laboratory test animal. Even morepreferably, the mammal is a human.

DETAILED DESCRIPTION

It is to be understood at the outset, that the figures and examplesprovided herein are to exemplify and not to limit the invention and itsvarious embodiments

As exemplified herein the inventors have for the first time identifiedligands of the epidermal growth factor receptor (EGFR) that are targetsof the miRNA miR-7 and are down-regulated in cancer cell lines by miR-7.Accordingly, provided in embodiments disclosed herein are methods andcompositions for the modulation of the expression and/or activity ofsuch EGFR ligands using miR-7, precursors and variants of miR-7, miRNAbearing the miR-7 seed region, and antagonists of such miRNA. Inparticular embodiments methods and compositions disclosed herein areused to treat diseases and conditions associated with dysregulation ofEGFR ligand expression or activity, such as cancer.

Embodiments of the invention employ, unless otherwise indicated,conventional molecular biology and pharmacology known to, and within theordinary skill of, those skilled the art. Such techniques are describedin, for example, “Molecular Cloning: A Laboratory Manual”, 2^(nd) Ed.,(ed. by Sambrook, Fritsch and Maniatis) (Cold Spring Harbor LaboratoryPress: 1989); “Nucleic Acid Hybridization”, (Hames & Higgins eds. 1984);“Oligonucleotide Synthesis” (Gait ed., 1984); Remington's PharmaceuticalSciences, 17th Edition, Mack Publishing Company, Easton, Pa., USA.; “TheMerck Index”, 12th Edition (1996), Therapeutic Category and BiologicalActivity Index,—and “Transcription & Translation”, (Hames & Higgins eds.1984).

Reference in this specification to any prior publication (or informationderived from it), or to any matter which is known, is not, and shouldnot be taken as, an acknowledgement or admission or any form ofsuggestion that prior publication (or information derived from it) orknown matter forms part of the common general knowledge in the field ofendeavour to which this specification relates,

miRNA

Micro RNAs (miRNAs) are small non-coding RNAs which function as,regulatory molecules in plants and animals to control gene expression bybinding complementary sites on mRNA. Without wishing to be bound by anytheory or hypothesis, the present invention is predicated on theinventors finding that the miRNA miR-7 specifically binds the 3′-UTR ofmRNA encoding EGFR ligands, especially transforming growth factor-alpha(TGF-α). Moreover, the inventors have surprisingly discovered thatincreasing the expression of miR-7 in cancer cells that express oroverexpress EGFR, such as head and neck cancer cells, results in areduced level of EGFR ligand mRNA and protein expression, G1 phase cellcycle arrest and cell death.

miRNAs bind the 3′UTRs of target mRNAs and important in this binding isa so-called ‘seed’ region of approximately 6-7 nucleotides near the 5′end of the miRNA (typically nucleotide positions 2 to 8). Accordingly,embodiments of the present invention broadly contemplate contactingcells or tissue, or administering to subjects in need thereof, one ormore miRNA, at least one of which comprises the seed region of miR-7. Inparticular embodiments this seed region comprises the sequence GGAAGA(SEQ ID NO:5).

In particular embodiments, miR-7 is employed. The nucleotide sequence ofhuman miR-7 is provided in SEQ ID NO:1. Additional sequence informationfor the miR-7 miRNA can be found athttp://microrna.sanger.ac.uk/sequences/index.shtml. Like most miRNAs,miR-7 is highly conserved between different species. Thus, whilsttypically the miRNA may be derived from the species of the subject to betreated, or constitute a sequence identical to miRNA from that species,this need not be the case in view of, for example, the high level ofsequence conservation of miRNA sequences between species.

Embodiments of the invention also contemplate the administration ofmiRNA variants of miR-7. Variants include nucleotide sequences that aresubstantially similar to sequences of miRNA disclosed herein. Variantsinclude nucleotide sequences that are substantially similar to sequencesof miRNA disclosed herein. In some embodiments, the variant miRNA to beadministered comprises a sequence displaying at least 80% sequenceidentity to the sequence of miR-7 (SEQ ID NO:1). In some embodiments,the miRNA to be administered comprises a sequence displaying at least90% sequence identity to SEQ ID NO:1. In other embodiments, the miRNA tobe administered comprises a sequence displaying at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1.Alternatively or in addition variants may comprise modifications, suchas non-natural residues at one or more positions with respect to themiR-7 sequence.

Also contemplated is the administration of a precursor molecule of miR-7or of a miRNA comprising a seed region comprising the sequence GGAAGA.miRNAs are generated from RNA precursors (pri-miRNAs) that usuallycontain several hundred nucleotides transcribed from regions ofnon-coding DNA. Pri-miRNAs are processed in the nucleus by RNase IIIendonuclease to form stem-loop precursors (pre-miRNAs) of approximately70 nucleotides. Pre-miRNAs are actively transported into the cytoplasmwhere they are further processed into short RNA duplexes, typically of21-23 bp, one of which represents the functional miRNA strand. Theadministration of such pri-miRNA and pre-miRNA precursors iscontemplated herein, wherein the pri-miRNA or pre-miRNA is cleaved andintracellularised to generate a functional miRNA.

In addition to the full-length miR-7 molecule, such as that shown in SEQID NO:1, the term “miR-7” also includes fragments of a miR-7 moleculeprovided the fragments are functional fragments. The term “fragment” ofa miRNA molecule means a portion of the full-length molecule. The sizeof the fragment is limited only in that it must be a functionalfragment, that is, able to modulate the expression of EGFR, modulatecell growth, and/or modulate cell differentiation. Typically, it willcomprise at least the seed region sequence GGAAGA (SEQ ID NO:5).

Administration of the miRNA may be directly to a subject in need oftreatment, or may be ex vivo administration to cells or tissue derivedfrom the subject. The miRNAs to be administered may be syntheticallyproduced or naturally derived from a cellular source.

Also contemplated by embodiments of the invention is the administrationof miRNAs linked to an additional agent capable of delivering the miRNAto the desired site. The additional agent may itself be capable ofinhibiting the activity and/or expression of an EGFR ligand. Forexample, miR-7 may be conjugated to an antibody directed to a cell typeknown to express an EGFR ligand in order to target the miR-7 to thosecells. In some embodiments the link between the miRNA and the additionalagent is a cleavable link. The presence of a cleavable link allows forcleavage of the miRNA from the additional agent for example afterinternalisation into cells expressing an EGFR ligand.

Also contemplated by embodiments of the invention is the administrationof agents capable of stimulating or enhancing the expression or activityof miRNA described herein. Such agents may be proteinaceous,non-proteinaceous or nucleic acid-based and include, for example,molecules and compounds capable of binding to the regulatory sequencesof miRNA genes to thereby induce or enhance the level of endogenousexpression of the miRNA. Those skilled in the art will appreciate thatthe scope of the invention is not so limited and any agents capable ofstimulating or enhancing miRNA expression or activity are contemplatedand fall within the scope of the present disclosure.

EGFR Ligands

The epidermal growth factor receptor (EGFR) family includes distincttyrosine kinase receptors, EGFR/HER/ErbB1, HER2/Neu/ErbB2, HER3/ErbB3and HER4/ErbB4. These receptors are widely expressed and are activatedby a family of at least twelve ligands that induce either homo- orhetero-dimerisation of the EGFR homologues. The ligands include membersof the epidermal growth factor family such as EGF, transforming growthfactor-alpha (TGFα), heparin binding EGF-like growth factor (HB-EGF),amphiregulin (AR), epiregulin (EPR), betacellulin (BTC), epigen and theneuregulins (NRG)-1, NRG-2, NRG-3 and NRG-4. In particular embodimentsof the invention the EGFR ligand, the expression or activity of which isto be modulated, is TGFα or HB-EGF, although the scope of the presentdisclosure is not so limited. More particularly: the EGFR ligand isTGFα.

In embodiments in which the subject is human, the 3′ UTR of TGFα mRNAtypically comprises the sequence provided in SEQ ID NO:12, or a variantthereof. Variants include nucleotide sequences that are substantiallysimilar to the sequence of SEQ ID NO:12. In some embodiments, thevariant 3′ UTR comprises a sequence displaying at least 80% sequenceidentity to the sequence of SEQ ID NO:12. In some embodiments, the 3′UTR comprises a sequence displaying at least 90% sequence identity toSEQ ID NO:12. In other embodiments, the 3′UTR comprises a sequencedisplaying at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:12.

As described herein the 3′ UTR of mRNA encoding EGFR ligands such asTGFα typically comprise one or more motifs or miR-7 target sites andparticular embodiments of the invention contemplate, the direct bindingof the miRNA employed to such sites in effecting the modulation of EGFRligand expression and/or activity. The 3′ UTR of the TGFα mRNA contains5 miR-7 target sites. The miR-7 target sites in the human TGFα 3′ UTRcomprise the sequences shown in SEQ ID Nos:7 to 11. Variants of suchtarget site sequences are also contemplated, including the generalisedor consensus miR-7 target site shown in SEQ ID NO:6. As for variants ofthe 3′ UTR sequence, miRNA target site variants may display at leastabout 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the miRNA target site sequences shown in SEQ ID Nos:6 to it

Antagonists

Embodiments of the invention also provide for the administration ofantagonists of the miRNA described herein in circumstances where it isdesirable to upregulate the expression and/or activity of the targetEGFR ligand. Those skilled in the art will readily appreciate thatsuitable antagonists for use in accordance with embodiments disclosedherein may take a variety of forms. The antagonist may be an antisenseconstruct comprising a nucleotide sequence specific to an miRNAdescribed herein, or a portion thereof, wherein the antisense constructinhibits, at least partially, the expression or activity of the miRNA.By “specific” it is meant that the antisense construct is substantiallyspecific for the miRNA, but not necessarily exclusively so. That is,while being specific for a particular miRNA sequence, the antisenseconstruct may also cross-hybridise with other sequences, such as othermiRNA sufficient to inhibit expression. Further, for example, thenucleotide sequence of an antisense construct according to the presentinvention may display less than 100% sequence identity with a particularmiRNA and retain specificity thereto. It will be appreciated by thoseskilled in the art that suitable antisense constructs need not binddirectly with the miRNA to which they are directed in order to effectthe expression or activity of those miRNA. Binding of an antisenseconstruct to its complementary cellular nucleotide sequence mayinterfere with transcription, RNA processing, transport, and/orstability of the miRNA to which it is specific. An antisense moleculemay comprise DNA, RNA, LNA, PNA or any combination thereof.

Suitable antisense constructs for use in accordance with embodimentsdisclosed herein include, for example, antisense oligonucleotides, smallinterfering RNAs (siRNAs) and catalytic antisense nucleic acidconstructs. Suitable antisense oligonucleotides may be prepared bymethods well known to those of skill in the art. Typicallyoligonucleotides will be chemically synthesized on automatedsynthesizers.

Those skilled in the art will readily appreciate that one or more basechanges may be made such that less than 100% complementarity existswhilst the oligonucleotide retains specificity for its miRNA and retainsantagonistic activity against this miRNA. Further, as described below,oligonucleotide sequences may include one or more chemical modificationswithout departing from the scope of the present invention.

Oligonucleotides in accordance with the invention may includemodifications designed to improve their delivery into cells, theirstability once inside a cell, and/or their binding to the appropriatemiRNA target. For example, the oligonucleotide sequence may be modifiedby the addition of one or more phosphorothioate (for examplephosphoromonothioate or phosphorodithioate) linkages between residues inthe sequence, or the inclusion of one or morpholine rings into thebackbone. Alternative non-phosphate linkages between residues includephosphonate, hydroxlamine, hydroxylhydrazinyl, amide and carbamatelinkages (see, for example, United States Patent Application PublicationNo. 20060287260, Manoharan I., the disclosure of which is incorporatedherein in its entirety), methylphosphonates, phosphorothiolates,phosphoramidates or boron derivatives. The nucleotide residues presentin the oligonucleotide may be naturally occurring nucleotides or may bemodified nucleotides. Suitable modified nucleotides include 2′-O-methylnucleotides, such as 2′-O-methyl adenine, 2′-O-methyl-uracil,2′-O-methyl-thymine, 2′-O-methyl-cytosine, 2′-O-methyl-guanine,2′-O-methyl-2-amino-adenine; 2-amino-adenine, 2-amino-purine, inosine;propynyl nucleotides such as 5-propynyl uracil and 5-propynyl cytosine;2-thio-thymidine; universal bases such as 5-nitro-indole; locked nucleicacid (LNA), and peptide nucleic acid (PNA). The ribose sugar moiety thatoccurs naturally in ribonucleosides may be replaced, for example with ahexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group asdescribed in United States Patent Application Publication No.20060035254, Manoharan et al., the disclosure of which is incorporatedherein in its entirety. Alternatively, or in addition, theoligonucleotide sequence may be conjugated to one or more suitablechemical moieties at one or both ends. For example, the oligonucleotidemay be conjugated to cholesterol via a suitable linkage such as ahydroxyprolinol linkage at the 3′ end.

Modified oligonucleotides with ‘silencing’ activity against specificmiRNA (“antagomirs”) are described in Krutzfeldt, J. et al., 2005,Nature 438:685-689, the disclosure of which is incorporated herein inits entirety are also antagonists of EGFR ligands. For example, anantagomir with sequence complementary to a miRNA specific for an EGFRligand may bind to the miRNA and this interaction inhibits the miRNA'sactivity. The antagomir may be 100% complementary to, for example, amiR-7 molecule or may be less than 100% complementary provided that theantisensemolecule is able to inhibit the function of miR-7. Antagomirsmay comprise 2-O-methyl nucleotides, phosphorothioate linkages betweenresidues at the 5′ and 3′ end, and a conjugated cholesterol moiety via ahydroxyprolinol linkage at the 3′ end. Embodiments as disclosed hereincontemplate use of antagomirs modified in the manner described inKrutzfeldt et al. as well as modifications or variations thereof. Thedesign of oligonucleotides or antagomirs for use in accordance withembodiments disclosed herein is well within the capabilities of thoseskilled in the art.

An alternative antisense technology is RNA interference (RNAi), see, eg.Chuang et al. (2000) PNAS USA 97: 4985) may be used to antagonise EGFRligands, according to known methods in the art (for example Fire et al.(1998) Nature 391: 806-811; Hammond, et al. (2001) Nature Rev, Genet. 2:110-1119; Hammond et al. (2000) Nature 404: 293-296; Bernstein et al(2001) Nature 409: 363-366; Elbashir et al (2001) Nature 411: 494-498;WO 99/49029 and WO 01/70949, the disclosures of which are incorporatedherein by reference), to inhibit the expression or activity of miRNA.RNAi refers to a means of selective post-transcriptional gene silencingby destruction of specific RNA by small interfering RNA molecules(siRNA). The siRNA is generated by cleavage of double stranded RNA,where one strand is identical to the message to be inactivated.Double-stranded RNA molecules may be synthesised in which one strand isidentical to a specific region of the miRNA transcript and introduceddirectly. Alternatively corresponding dsDNA can be employed, which, oncepresented intracellularly is converted into dsRNA. Methods for thesynthesis of suitable molecules for use in RNAi and for achievingpost-transcriptional gene silencing are known to those of skill in theart.

A further means of inhibiting the expression or activity of miRNAincludes introducing catalytic antisense nucleic acid constructs, suchas DNAzymes and ribozymes, which are capable of cleaving miRNAtranscripts. Ribozymes, for example, are targeted to, and anneal with, aparticular sequence by virtue of two regions of sequence complementarityto the target flanking the ribozyme catalytic site. After binding, theribozyme cleaves the target in a site-specific manner. The design andtesting of ribozymes which specifically recognise and cleave miRNAsequences can be achieved by techniques well known to those in the art(for example Lieber and Strauss, (1995) Mol. Cell. Biol. 15:540-551, thedisclosure of which is incorporated herein by reference).

Antibodies against endogenous miRNAs may also be antagonists. The term“antibody” includes within its meaning anti-miR-7 polyclonal andmonoclonal antibodies (including agonist, antagonist, and neutralizingantibodies), anti-miR-7 antibody compositions with polyepitopicspecificity, single chain anti-miRNA antibodies, and fragments ofanti-miRNA antibodies. The term “monoclonal antibody” as used hereinrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Antibody fragmentscomprise a portion of an intact antibody, preferably the antigen bindingor variable region of the intact antibody. Examples of antibodyfragments include Fab, Fab′, F(abs)₂, and Fv fragments; diabodies,linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

It will also be recognised by those skilled in the art that anantagonist in accordance with embodiments of the invention may effect amodulator or regulator of the expression or activity of a miRNAdisclosed herein. Similarly, the antagonist may affect a target of amiRNA disclosed herein. Thus, antagonists may take any suitable form,depending on the nature and identity of the molecule(s) to be effected,such as for example a small molecule inhibitor, peptide inhibitor orantibody.

Methods of Modulating EGFR Ligand Expression and/or Activity

In particular embodiments the present invention provides methods ofmodulating the expression of an EGFR ligand in a cell or tissue bycontacting the cell or tissue with an miRNA as disclosed herein, or anantagonist of such an miRNA. Examples of cells which express EGFRligands include cancer cells, lung cells, bone cells, blood cells, andskin cells. The cell may be isolated or purified from a subject, may belocated in a sample from a subject, or may be located in or on asubject. Typically the EGFR ligand expression and/or activity isdecreased in the cell or tissue following contact of the cell or tissuewith a miRNA as disclosed herein compared to the level in a cell ortissue which has not been contacted with the miRNA. Similarly, theexpression and/or activity of the EGFR ligand is typically increased inthe cell or tissue following contact of the cell or tissue with anantagonist of a miRNA as disclosed herein compared to the level in asample of a subject which has not been contacted with the antagonist.

Contacting the cell or tissue with the miRNA or antagonist may beachieved by any method known in the art. In some embodiments the cellhas been isolated from the subject and combining the cell and the miRNAor antagonist thereof occurs ex vivo or in vitro. In other embodimentsthe cell has not been isolated from the subject and contacting the celland the miRNA or antagonist thereof occurs in vivo. The miRNA orantagonist may be contacted with the cell directly, i.e. applieddirectly to a cell requiring modulation of EGFR ligand expression oractivity, or alternatively may be combined with the cell indirectly,e.g. by injecting the molecule into the bloodstream of a subject, whichthen carries the molecule to the cell requiring modulation of EGFRligand expression or activity. Further, a sample may be removed from asubject and combined with an miRNA or antagonist in vitro prior toreturning at least a portion of the sample back to the subject. Forexample, the sample may be a blood sample which is removed from asubject and combined with the molecule prior to injecting at least aportion of the blood back into the subject.

In some embodiments the miRNA or antagonist thereof is contacted with acell, wherein the endogenous levels of the miRNA are different ascompared to the cell before contacting with the miRNA or antagonist. Theterm, “endogenous” as used in this context refers to the“naturally-occurring” levels of expression and/or activity of therelevant miRNA. In these embodiments, compounds or compositions can becontacted with cells such that the expression and/or activity of themiRNA are increased or decreased as compared to the“naturally-occurring” levels.

In some embodiments administration of polynucleotides (miRNA ornucleic-acid based antagonists thereof) is via a vector (e.g.viral)-based approach, or by administration of a polynucleotide in theform of a fusion protein where the polynucleotide is bound to aprotamine-Fab antibody fragment which targets the polynucleotide tocells of interest, i.e. cells expressing EGFR ligands.

Diseases and Conditions

EGFR ligands are dysregulated in many conditions including cancer.Accordingly, methods and compositions provided herein for modulating theexpression and/or activity of an EGFR ligand using antagonists asdescribed above are also applicable to the treatment or prevention ofconditions associated with EGFR ligand dysregulation. Conditions towhich methods and compositions of the invention are applicable include,but are not limited to cancer, renal disease, pulmonary disease, cardiacdisease, skin disease or infectious disease. The term “cancer” as usedherein refers to any malignant cell growth or tumour caused by abnormaland uncontrolled cell division.

The cancer may be any cancer in which the expression or activity of anEGFR ligand, such as TGFα, capable of being modulated by an miRNA asdescribed herein (or antagonist thereof) is dysregulated. Typically suchcancers will be associated with upregulated or elevated levels ofexpression or activity of the EGFR ligand relative to normal cells andtissues. Exemplary cancers include, but are not limited to liver,ovarian, colorectal, lung, small cell lung, breast, prostate,pancreatic, renal, colon, gastric, endometrial, stomach, oesophageal,and head and neck cancers, peritoneal carcinomatosis, lymphoma, sarcomaor secondary metastases thereof, glioblastoma, neuroblastoma, andmelanoma.

Compositions and Routes of Administration

Embodiments of the present invention contemplate compositions formodulating the expression and/or activity of an EGFR ligand in a cell,tissue or subject and for treating or preventing a condition associatedwith dysregulation an EGFR ligand. Such compositions may be administeredin any convenient or suitable route such as by parenteral (including,for example, intraarterial, intravenous, intramuscular, subcutaneous),oral, nasal, mucosal (including sublingual), intracavitary or topicalroutes. Thus compositions may be formulated in a variety of formsincluding solutions, suspensions, emulsions, and solid forms and aretypically formulated so as to be suitable for the chosen route ofadministration, for example as capsules, tablets, caplets, elixirs fororal ingestion, in an aerosol form suitable for administration byinhalation (such as by intranasal inhalation or oral inhalation),ointment, cream, gel, jelly or lotion suitable for topicaladministration, or in an injectible formulation suitable for parenteraladministration. The preferred route of administration will depend on anumber of factors including the condition to be treated and the desiredoutcome. The most advantageous route for any given circumstance can bedetermined by those skilled in the art. For example, in circumstanceswhere it is required that appropriate concentrations of the desiredagent are delivered directly to the site in the body tote treated,administration may be regional rather than systemic. Regionaladministration provides the capability of delivering very high localconcentrations of the desired agent to the required site and thus issuitable for achieving the desired therapeutic or preventative effectwhilst avoiding exposure of other organs of the body to the compound andthereby potentially reducing side effects.

In general, suitable compositions may be prepared according to methodswhich are known to those of ordinary skill in the art and may include apharmaceutically acceptable diluent, adjuvant and/or excipient. Thediluents, adjuvants and excipients must be “acceptable” in terms ofbeing compatible with the other ingredients of the composition, and notdeleterious to the recipient thereof.

Examples of pharmaceutically acceptable diluents are demineralised ordistilled water; saline solution; vegetable based oils such as peanutoil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oilssuch as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil,sesame oil, arachis oil or coconut oil; silicone oils, includingpolysiloxanes, such as methyl polysiloxane, phenyl polysiloxane andmethylphenyl polysolpoxane; volatile silicones; mineral oils such asliquid paraffin, soft paraffin or squalane; cellulose derivatives suchas methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols,for example ethanol or iso-propanol; lower aralkanols; lowerpolyalkylene glycols or lower alkylene glycols, for example polyethyleneglycol, polypropylene glycol, ethylene glycol, propylene glycol,1,3-butylene glycol or glycerin; fatty acid esters such as isopropylpalmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone;agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly.Typically, the carrier or carriers will form from 1% to 99.9% by weightof the compositions.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, medium chain triglyceride (MCT), isotonic saline, phosphatebuffered saline, ethanol and 1,2 propylene glycol. Some examples ofsuitable carriers, diluents, excipients and adjuvants for oral useinclude peanut oil, liquid paraffin, sodium carboxymethylcellulose,methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose,sucrose, sorbitol, mannitol, gelatine and lecithin. In addition theseoral formulations may contain suitable flavouring and colourings agents.When used in capsule form the capsules may be coated with compounds suchas glyceryl monostearate or glyceryl distearate which delaydisintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice, sweeteners, disintegratingagents, diluents, flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthangum, bentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, flavouring agents include peppermint oil, oil ofwintergreen, cherry, orange or raspberry flavouring. Suitable coatingagents include polymers or copolymers of acrylic acid and/or methacrylicacid and/or their esters, waxes, fatty alcohols, zein, shellac orgluten. Suitable preservatives include sodium benzoate, vitamin E,alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben orsodium bisulphite. Suitable lubricants include magnesium stearate,stearic acid, sodium oleate, sodium chloride or talc. Suitable timedelay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents. Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acid,polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

Emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as guar gum, gum acaciaor gum tragacanth.

Methods for preparing parenterally administrable compositions areapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein. The composition may incorporate any suitable surfactant such asan anionic, cationic or non-ionic surfactant such as sorbitan esters orpolyoxyethylene derivatives thereof. Suspending agents such as naturalgums, cellulose derivatives or inorganic materials such as silicaceoussilicas, and other ingredients such as lanolin, may also be included.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins,Pennsylvania, USA. The carrier will depend on the route ofadministration, and again the person skilled in the art will readily beable to determine the most suitable formulation for each particularcase.

The compositions may also be administered in the form of liposomes.Liposomes are generally derived from phospholipids or other lipidsubstances, and are formed by mono- or multilamellar hydrated liquidcrystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes can be used. The compositions in liposome form may containstabilisers, preservatives, excipients and the like. The preferredlipids are the phospholipids and the phosphatidyl cholines (lecithins),both natural and synthetic. Methods to form liposomes are known in theart, and in relation to this specific reference is made to: Prescott,Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 etseq., the contents of which is incorporated herein byreference.

Combination Regimens

Therapeutic advantages may be realised through combination regimens. Incombination therapy the miRNA, antagonist thereof, or agent capable ofstimulating or enhancing the expression or activity of the miRNA and atleast an additional therapeutic agent may be coadministered. Forexample, in the context of cancer, one may seek to maintain ongoinganti-cancer therapies such as chemotherapy and/or radiotherapy, in orderto manage the condition of the patient, to improve local tumour controland/or reduce the risk of metastasis, whilst employing agents inaccordance with embodiments of the present invention. Accordingly,methods of treatment according to the present invention may be appliedin conjunction with conventional therapy, such as with tyrosine kinaseinhibitors, radiotherapy, chemotherapy, surgery, or other forms ofmedical intervention. By “coadministered” is meant simultaneousadministration in the same formulation or in two different formulationsvia the same or different routes or sequential administration by thesame or different routes. By “sequential” administration is meant atime, difference of, for example, from seconds, minutes, hours, days,weeks or months between the administration of the two formulations ortherapies. The formulations or therapies may be administered in anyorder.

The additional therapeutic agent(s) used will depend upon the conditionto be treated or prevented. For example where the condition is a headand neck cancer, suitable therapeutic agents include erlotinib(Tarceva), gefitinib (Iressa or ZD1839) or cetuximab. Alternatively orin addition, the antagonist such as the miRNA may be administeredsimultaneously and/or consecutively in any order with an agent whichcounters the side effects of the miRNA.

Examples of chemotherapeutic agents include adriamycin, taxol,fluorouracil, melphalan, cisplatin, oxaliplatin, alpha interferon,vincristine, vinblastine, angioinhibins, TNP-470, pentosan poiysulfate,platelet factor 4, angiostatin, LM-609, SU-101, CM-101, Techgalan,thalidomide, SP-PG and the like. Other chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates including busulfan; triazines including dicarbazine;ethyenimines including thiotepa and hexamethylmelamine; folic acidanalogues including methotrexate; pyrimidine analogues including5-fluorouracil, cytosine arabinoside; purine analogues including6-mercaptopurine and 6-thioguanine; antitumour antibiotics includingactinomycin D; the anthracyclines including doxorubicin, bleomycin,mitomycin C and methramycin; hormones and hormone antagonists includingtamoxifen and cortiosteroids and miscellaneous agents includingcisplatin and brequinar, and regimens such as COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine anddexamethasone), and PROMACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, taxol,etoposide/mechlorethamine, vincristine, prednisone and procarbazine).

Agents and compositions disclosed herein may be administeredtherapeutically or preventively. In a therapeutic application, agentsand compositions are administered to a patient already suffering from acondition, in an amount sufficient to cure or at least partially arrestthe condition and its symptoms and/or complications. The agent orcomposition should provide a quantity of the active compound sufficientto effectively treat the patient.

Dosage

The effective dose level of the administered agent for any particularsubject will depend upon a variety of factors including: the type ofcondition being treated and the stage of the condition; the activity andnature of the agent employed; the composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of sequestrationof compounds; the duration of the treatment; drugs used in combinationor coincidental with the treatment, together with other related factorswell known in medicine. One skilled in the art would be able, by routineexperimentation, to determine an effective, non-toxic dosage which wouldbe required to treat applicable conditions. These will most often bedetermined on a case-by-case basis.

Generally, an effective dosage is expected to be in the range of about0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically,about 0.001 mg to about 750 mg per kg body weight per 24 hours; about0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg toabout 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; or about 1.0 mg to about 250 mg perkg body weight per 24 hours. More typically, an effective dose range isexpected to be in the range of about 10 mg to about 200 mg per kg bodyweight per 24 hours.

Alternatively, an effective dosage may be up to about 5000 mg/m².Generally, an effective dosage is expected to be in the range of about10 to about 5000 mg/m², typically about 10 to about 2500 mg/m², about 25to about 2000 mg/m², about 50 to about 1500 mg/m², about 50 to about1000 mg/m², or about 75 to about 600 mg/m². Further, it will be apparentto one of ordinary skill in the art that the optimal quantity andspacing of individual dosages will be determined by the nature andextent of the condition being treated, the form, route and site ofadministration, and the nature of the particular individual beingtreated. Also, such optimum conditions can be determined by conventionaltechniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of thecomposition given per day for a defined number of days, can beascertained by those skilled in the art using conventional course oftreatment determination tests.

In some embodiments effective dosages, optimal number of dosages,spacing of individual dosages and optimal courses of treatment may bedetermined by monitoring serum or plasma levels of an EGFR ligand. Forexample, a sample such as a blood serum or blood plasma, may be assayedby any method known in the art to determine the level of expressionand/or activity of the EGFR ligand. After administration of the agent orat intervals during the course of treatment a further sample may betaken and assayed to determine the level of expression and/or activityof the EGFR ligands. In instances where the levels expression and/oractivity of the EGFR ligand has not changed significantly the dose orfrequency of doses may increased to optimise the dosage or thetreatment. In instances where the lever of EGFR ligands have changedsignificantly the dose or frequency of doses may be decreased tooptimise the dosage or treatment.

The efficacy of a treatment regime in a subject suffering from a diseaseor condition associated with dysregulated expression or activity of anEGFR ligand may be evaluated by monitoring the change in expression ofan EGFR ligand in the subject. For example, a subject may be treatedwith a miR-7 miRNA, a precursor or variant thereof, a miRNA comprising aseed region comprising the sequence GGAAGA, or an antagonist of any suchmiRNA. After a first period of time a biological sample from the subjectmay be assayed by any method known in the art to determine the level ofexpression and/or activity of the EGFR ligand in the sample. After afurther period of time an additional biological sample from the subjectmay be assayed by any method known in the art to determine the level ofexpression and/or activity of the EGFR ligand in the additional sample.In some embodiments this process of sampling and determining EGFR ligandlevels may be repeated at more than two intervals such that the level ofEGFR ligand in response to the treatment regime can be measured overtime. The efficacy of the regime can then be evaluated by determiningwhether the expression and/or activity of the EGFR ligand changes overthe period of time. A change in the level of expression and/or activityof the EGFR ligand is indicative of the efficacy of the treatmentregime.

The EGFR ligand may be TGFa, HB-EGF, amphiregulin, epiregulin,betacellulin, epigen NRG-1, NRG-2, NRG-3 or NRG-4. In particularembodiments the EGFR ligand may be TGFa.

The present invention will now be further described in greater detail byreference to the following specific examples, which should not beconstrued as in any way limiting the scope of the invention.

EXAMPLES Example 1 cDNA Microarray Expression Profiling and DataAnalysis

Microarray analysis was used to identify novel genes down-regulated bymiR-7. Specifically, cDNA microarray analysis of miR-7 transfected HN5cells revealed new miR-7 targets with potential roles in HNC. HN5 cellswere transfected for 24 h with a miR-NC precursor corresponding to humanmiR-7 (Pre-miR miRNA Precursor Product ID: PM10047) (Ambion; Victoria,Australia) or a negative control miRNA (miR-NC; Pre-miR miRNA PrecursorNegative Control #1, Product ID: AM17110) (Ambion; Victoria, Australia).Total RNA was isolated for microarray analysis. The 24 h time point wasselected on the basis of previous studies which identified a number ofmiRNA-regulated genes in a liver cancer and non small cell lung cancercell line (Wang & Wang (2006), Nucleic Acids Res 34:1646-1652; Websteret al., 2009, J Biol Chem 284:5731-5741).

Total RNA was isolated from HN5 cells 24 h after transfection (6 wellplates seeded at a density of 5.0×10⁵ cells per well) with miR-7 ormiR-NC precursor molecules (30 nM) using TRizol reagent (Invitrogen;Victoria, Australia). The quantity and integrity of extracted RNA wasconfirmed using a 2100 Bioanalyzer (Agilent Technologies; Victoria,Australia) before samples were judged suitable for array analysis. Geneexpression profiling by microarray hybridisation was performed with twoexperimental replicates by the Australian Genome Research Facility(Victoria, Australia) using Human-6 v3 array chips (Illumina; Victoria,Australia). Raw data, consisting of genes significantly up ordown-regulated (p<0.05) in response to transfection with miR-7 precursorby at least 1.5-fold relative to miR-NC precursor, was generated usingthe Database for annotation, visualisation and integrated discovery(DAVID) (Dennis et al., (2003), Genome Biol 4:P3; Huang et al., (2009),Nat Protoc 4:44-57). This was followed by further DAVID analysis of thedown-regulated gene list for identification of signalling pathwaysenriched for molecules down-regulated by miR-7. DIANA-mirExTra was usedto confirm the over-representation of putative miR-7 target genes amongthe microarray list of genes down-regulated by miR-7. TargetScan (Lewiset al., (2005), Cell 120:15-20) was used for miR-7 target predictionswithin signalling pathways enriched for putative miR-7 target genes.

Total RNA was extracted from HN5 cells with TRIzol reagent (Invitrogen;Victoria, Australia) and treated with DNase I (Promega; Sydney,Australia) to eliminate contaminating genomic DNA. For qRT-PCR analysisof EGFR, RAF1, PAK1 and GAPDH mRNA expression, 0.5 μg of total RNA wasreverse transcribed into cDNA with random hexamers using Thermoscript(Invitrogen; Victoria, Australia). Real-time PCR for EGFR, RAF1, PAK1and GAPDH cDNA was performed on a. Corbett 3000 RotorGene instrument(Corbett Research; Sydney, Australia) using a SensiMixPlus SYBR Kit(Quantace; New South Wales, Australia) and EGFR, RAF1, PAK1 and GAPDHprimers from PrimerBank (Wang & Seed, 2003, Nucleic Acid Res., 31:e154): EGFR-F, 5′-GCG TTC GGC ACG GTG TAT AA-3′ (SEQ ID NO:13); EGFR-R,5′-GGC TTT CGG AGA TGT TGC TTC-3′ (SEQ ID NO:14); RAF1-F, 5′-GCA CTG TAGCAC CAA AGT ACC-3′ (SEQ ID NO:15); RAF1-R, 5′-CTG GGA CTC CAC TAT CACCAA TA-3′ (SEQ ID NO:16); PAK1-F, 5′-CAG CAC TAT. GAT TGG AGT CGG-3′(SEQ ID NO:17); PAK1-R, 5′-TGG ATC GGT AAA ATC GGT CCT-3′ (SEQ IDNO:18); GAPDH-F, 5′-ATG GGG MG G-TG MG GTC G-3′ (SEQ ID NO:19); GAPDH-R,5′-GGG GTC ATT GAT GGC AAC ATT A-3′ (SEQ ID NO:20). Single peak meltcurves and reaction efficiencies of >0.9 were required for furtheranalysis of data. Expression of EGFR, RAF1 and PAK1 mRNA relative toGAPDH mRNA was determined using the 2^(−ΔΔCT) method (Livak & Schmittgen(2001), Methods 25:402-408).

All results are presented as means±standard deviation (S.D.).Statistical significance was calculated using Student's t test(two-tailed, unpaired) and the level of significance was set at p<0.05.All samples for immunoblotting were loaded in duplicate to validateequal loading of protein. Statistical analysis of qRT-PCR data wasperformed using GenEx software (MultiD; California, USA). Normality ofdata was confirmed using the Kolmogorov-Smimov test (KS test).

Two experimental replicates for each treatment, miR-7 precursor ormiR-NC precursor, were analysed by microarray. The microarray analysisidentified 189 genes that were significantly down-regulated (p<0.05) byat least 1.5-fold in the miR-7-transfected cells relative to themiR-NC-transfected cells (data not shown). DAVID analysis of thedown-regulated genes identified by microarray analysis revealed thatmiR-7 targets a variety of molecules belonging to the ErbB receptorsignalling pathway, with this molecular pathway having the greatestfold-enrichment (7.2-fold) for miR-7 down-regulated genes (p<0.001). Thetop six genes belonging to the ErbB receptor signalling pathway thatwere most down-regulated following miR-7 transfection are presented inTable 1 and confirmed that miR-7 was able to down-regulate multiplegenes from the EGFR signalling pathway, genes previously unidentified asmiR-7 targets in HNC. DIANA mirExTra was used to investigate whetherthere was enrichment for predicted miR-7 targets within thedown-regulated genes. This analysis revealed that 135 of 189down-regulated genes were putative miR-7 targets (p<0.001), validating,the microarray approach to identify genes down-regulated by miR-7, as itwas hypothesised that a significant proportion of the genesdown-regulated by miR-7 would contain miR-7 target sites.

TABLE 1 Gene Symbol Number of putative (common name) Fold Change p-valuemiR-7 target sites EGFR (EGFR) ↓3.27 1.89 × 10⁻³ 3 RAF1 (RAF1) ↓2.658.21 × 10⁻³ 2 TGFA (TGFα) ↓2.16 2.49 × 10⁻² 5 PIK3CD (PI3K) ↓2.03 9.36 ×10⁻³ 4 ELK1 (ELK1) ↓1.86 4.78 × 10⁻³ 0 PAK1 (PAK1) ↓1.81 9.03 × 10⁻³ 1HBEGF (HB-EGF) ↓1.50 2.75 × 10⁻² 0

Of the genes down-regulated by miR-7 in the microarray, RAF1 and PAK1(Table 1) were experimentally confirmed using qRT-PCR. These were chosenbecause of their known role in EGFR signaling (RAF1) and Akt activation(PAK1) in other cancers and normal tissues. qRT-PCR validation of RAF1and PAK1 as miR-7 targets was performed using RNA from HN5 cellstransfected with miR-7 precursor or miR-NC precursor and it wasconfirmed that RAF1, and PAK1 mRNA was significantly down-regulatedrelative to GAPDH mRNA in samples transfected with miR-7 (FIG. 1). RAF1mRNA was down-regulated 2.49-fold (p<0.001) and PAK1 mRNA wasdown-regulated 1.82-fold (p<0.01) (FIG. 1), thus experimentallyconfirming that these genes are targets of miR-7 and suggesting thatmiR-7 promotes decay of RAF1 and PAK1 mRNA in HN5 cells.

As noted above, DAVID analysis of the down-regulated microarray genesidentified the ErbB signalling pathway as that most enriched for genesdown-regulated by miR-7. A schematic representation (FIG. 2) shows thepossible interactions between these genes and miR-7. It is apparent thatmiR-7 has the capacity to regulate the EGFR signalling pathway miR-7down-regulates multiple members of the EGFR signalling pathway in HN5HNC cells. Genes significantly changing in expression by microarrayanalysis in response to miR-7 treatment vs. negative control are listed,and the fold changes, p-values and number of putative miR-7 target sitesare indicated. The presence of putative miR-7 target sites within the3′-UTR of a gene indicates that this gene is possibly a direct target ofmiR-7, whereas the lack of putative miR-7 target sites within the 3′-UTRof a gene indicates that this gene is possibly an indirect target ofmiR-7.

The observed down-regulation of EGFR following miR-7 transfection was inaccordance with other experimental findings and served to validate themicroarray analysis. Interestingly, two EGFR-activating ligands withinthe ErbB signalling pathway, TGFα and HB-EGF, were down-regulated in themicroarray, both previously unidentified potential targets of miR-7.TGFα and HB-EGF are commonly over-expressed in cancers, including HNCs,and have been shown to contribute to increased proliferation of HNCcells (Grandis et al., (2008), J Cell Biochem 69:55-62). Thusdown-regulation of these ligands indicates that miR-7 is able to disruptEGFR signalling at the ligand and receptor levels as well as disruptingautocrine loops in order to down-regulate the EGFR signalling pathwayand reduce tumour growth. It has also been shown that in colorectal,rectal and epidermoid carcinoma cell lines treated with increasingconcentrations of cetuximab, a monoclonal antibody which blocks bindingof ligands to EGFR, there is a dose dependent increase in concentrationof TGFα in serum (Mutsaers et al., (2009), Clin Cancer Res15:2397-2405). This suggests that blocking of TGFα binding results inupregulation of ligand production in these cancers. Furthermore, it hasbeen found that TGFa and EGFR mRNA expression is significantly increasedin both HNC tumour tissue and surrounding histologically normal tissue,which could lead to malignant transformation of normal tissue (Grandis &Tweardy (1993), J Cell Biochem Suppl 17F:188-191). This reinforces thenotion that down-regulation of EGFR ligands by miR-7 results indecreased EGFR pathway signalling and tumour growth and aids inpreventing HNC recurrence in patients.

Example 2 miR-7 Modulates TGFα

The inventors then investigated the ability of miR-7 to directlymodulate the level of expression of TGFα in HNC cell lines. Thefollowing DNA plasmids were used: pRL-CMV Renilla luciferase reporter(Promega) and pGL3-consensus miR-7 target site (SEQ ID NO:6) fireflyluciferase plasmid (Webster et al., 2009). pGL3-TGFα miR-7 target sitenumber 5 (SEQ ID NO:11) was generated by ligating annealed DNAoligonucleotides corresponding to nt 3699-3751 (SEQ ID NO:21) of theTGFα mRNA 3′-UTR (GenBank accession number NM_(—)003236.2) into uniqueSpeI and ApaI sites that were inserted 3′ of the luciferase open readingframe of pGL3-control (Promega) firefly luciferase reporter vector. Thesequence of all plasmids was confirmed by sequencing.

The HNC cell line FaDu was obtained from the American Type CultureCollection (ATCC) and HNC cell line HN5 was kindly provided by A/Prof.Terrance Johns (Monash Institute of Medical Research). FaDu and HN5 celllines were cultured at 37° C. in 5% CO₂ in low glucose DMEM (Invitrogen)supplemented with 10% foetal bovine serum (FBS). Cell lines were usedwithin 20 passages of initial stock for all experiments. For analysis ofbasal EGFR pathway expression and signaling, cells were seeded in 6 wellplates at a density ranging from 2.8-4.0×10⁵ cells per well, and 24 hafter plating were serum starved for 24 h in DMEM supplemented with 0.5%FBS prior to protein extraction.

Cells were seeded at a density of 4.5×10⁵ (FaDu) or 5.0×10⁵ (HN5) cellsin 6 well plates and transfected using Lipofectamine 2000 (Invitrogen)with miR-7 or miR-NC precursor molecules at final concentrations rangingfrom 1-30 nM. Cells were harvested at 24 h for RNA extraction or 3 d forprotein extraction.

For quantitative reverse transcription PCR analysis total RNA wasextracted from HN5 cells with TRIzol reagent (Invitrogen) and treatedwith DNase I (Promega) to eliminate contaminating genomic DNA. ForqRT-PCR analysis of TGFα and GAPDH mRNA expression, 0.5 μg of total RNAwas reverse transcribed into cDNA with random hexamers usingThermoscript (Invitrogen). Real-time PCR for TGFα and GAPDH cDNA wasperformed on a Corbett 3000 RotorGene instrument (Corbett Research)using a SensiMixPlus SYBR Kit (Quantace) and TGFα and GAPDH primers fromPrimerBank (Wang and Seed, 2003): TGFα-F, 5′-TGT MT CAC CTG TGC AGC CTTT-3′ (SEQ ID NO:22); TGFα-R, 5′-GTG GTC CGC TGA TTT CTT CTC T-3′ (SEQ IDNO:23); GAPDH-F, 5′-ATG GGG MG GTG MG GTC G-3′ (SEQ ID NO:19); GAPDH-R,5′-GGG GTC ATT GAT GGC MC ATT A-3′ (SEQ ID NO:20). Single peak meltcurves and reaction efficiencies of >0.9 were required for furtheranalysis of data. Expression of TGFα mRNA relative to GAPDH mRNA wasdetermined using the 2^(−ΔΔCT) method (Livak and Schmittgen, 2001).

For luciferase reporter assays, cells were seeded at a density of2.0×10⁵ cells per well in 24 well plates and co-transfected usingLipofectamine 2000 (Invitrogen) with miR-7 or miR-NC precursor molecules(0.5-1 nM), and 100 ng per well of firefly luciferase reporter DNA and 5ng per well of pRL-CMV Renilla luciferase reporter as a transfectioncontrol. Lysates were collected 24 h after transfection using 1× PassiveLysis Buffer (Promega), frozen at −80° C. overnight, thawed andcentrifuged at 13,000×g for 5 min. Each supernatant was assayed forfirefly and Renilla luciferase activity using a Dual-Luciferase ReporterAssay System (Promega) and a FLUOstar OPTIMA luminometer (BMG Labtech).Relative luciferase expression was determined by normalising fireflyluciferase values to Renilla luciferase values.

All results are presented as means±standard deviation (S.D.).Statistical significance was calculated using Student's t test(two-tailed, unpaired) and the level of significance was set at p<0.05.All samples for immunoblotting were loaded in duplicate to validateequal loading of protein. Statistical analysis of qRT-PCR data wasperformed using GenEx software (MultiD). Normality of data was confirmedusing the Kolmogorov-Smimov test (KS test).

As shown in FIG. 3, expression of TGFα was significantly reduced both inHN5 and FaDu cells in the presence of miR-7, a reduction of 3.3 fold(p-value−6.61×10⁻⁴) relative to miR-NC in HN5 cells and a reduction of1.41 fold (p-value−7.43×10⁴) relative to miR-NC in FaDu cells. Theresults of the luciferase assays (FIG. 4) illustrate that in HN5 cellsmiR-7 binds to both a consensus miR-7 binding motif and the predictedmiR-7 binding motif 5 provided in SEQ ID NO:11.

1. A method for modulating the expression and/or activity of anepidermal growth factor receptor (EGFR) ligand in a cell or tissue, themethod comprising contacting the cell or tissue with a miR-7 miRNA, aprecursor or variant thereof, a miRNA comprising a seed regioncomprising the sequence GGAAGA, or an antagonist of any such miRNA. 2.(canceled)
 3. The method of claim 1 wherein the miR-7 miRNA comprisesthe nucleotide sequence set forth in SEQ ID NO:1.
 4. (canceled)
 5. Themethod of claim 1 wherein the miR-7 miRNA precursor comprises a sequenceas set forth in any one of SEQ ID Nos:2 to
 4. 6. The method of claim 1wherein contacting the cell or tissue with the miRNA reduces or inhibitsthe expression and/or activity of the EGFR ligand.
 7. The method ofclaim 1 wherein contacting the cell or tissue with an antagonist of themiRNA increases the expression and/or activity of the EGFR ligand. 8.The method of claim 1 wherein the 3′ untranslated region of the mRNAencoding the EGFR ligand comprises one or more miRNA binding sites. 9.The method of claim 8 wherein the miRNA binding sites comprise sequencesas set forth in any of SEQ ID Nos:6 to
 11. 10. The method of claim 1wherein the EGFR ligand is TGFα or HB-EGF.
 11. (canceled)
 12. The methodof claim 10 wherein the mRNA encoding TGFα comprises a 3′ untranslatedregion comprising the sequence set forth in SEQ ID NO:12, or a variantthereof. 13-14. (canceled)
 15. The method of claim 1 wherein a subjectcontaining the cell or tissue, or from which the cell or tissue isderived, suffers from, is predisposed to, or is otherwise at risk ofdeveloping a disease or condition associated with dysregulatedexpression or activity of the EGFR ligand.
 16. The method of claim 15wherein the disease or condition is a cancer.
 17. A method for treatinga disease or condition associated with dysregulated expression oractivity of an EGFR ligand in a subject, comprising administering to thesubject an effective amount of a miR-7 miRNA, a precursor or variantthereof, a miRNA comprising a seed region comprising the sequenceGGAAGA, or an antagonist of any such miRNA, whereby the miRNA modulatesthe expression and/or activity of the EGFR ligand.
 18. The method ofclaim 17 wherein the disease or condition is associated with upregulatedor elevated expression or activity of the EGFR ligand and the subject isadministered an effective amount of a miR-7 miRNA, a precursor orvariant thereof, a miRNA comprising a seed region comprising thesequence GGAAGA.
 19. (canceled)
 20. The method of claim 17 wherein themiR-7 miRNA comprises the nucleotide sequence set forth in SEQ ID NO:1.21. (canceled)
 22. The method of claim 17 wherein the miR-7 miRNAprecursor comprises a sequence as set forth in any one of SEQ ID Nos:2to
 4. 23. The method of claim 17 wherein the 3′ untranslated region ofthe mRNA encoding the EGFR ligand comprises one or more miRNA bindingsites.
 24. The method of claim 23 wherein the miRNA binding sitescomprise sequences as set forth in any of SEQ ID Nos:6 to
 11. 25. Themethod of claim 17 wherein the EGFR ligand is TGFα or HB-EGF. 26.(canceled)
 27. The method of claim 25 wherein the mRNA encoding TGFαcomprises a 3′ untranslated region comprising the sequence set forth inSEQ ID NO:12, or a variant thereof.
 28. The method of claim 17 whereinthe disease or condition is a cancer.
 29. The method of claim 28 whereinthe cancer is selected from the group consisting of head and neckcancer, glioblastoma, pancreatic cancer, colon cancer, lung cancer,prostate cancer, breast cancer, liver cancer, neuroblastoma andmelanoma. 30-32. (canceled)
 33. A method for preventing or reducingtumour growth, cancer metastasis or reoccurrence in a subject, whereinthe tumour or cancer is associated with upregulated or elevatedexpression or activity of an EGFR ligand, the method comprisingadministering to the subject an effective amount of a miR-7 miRNA, aprecursor or variant thereof, a miRNA comprising a seed regioncomprising the sequence GGAAGA, whereby the miRNA modulates theexpression and/or activity of the EGFR ligand.